Mitochondrial oxidative stress promotes atrial fibrillation

Oxidative stress has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). Indeed, the prevalence of AF increases with age as does oxidative stress. However, the mechanisms linking redox state to AF are not well understood. In this study we identify a link between oxidative stress and aberrant intracellular Ca2+ release via the type 2 ryanodine receptor (RyR2) that promotes AF. We show that RyR2 are oxidized in the atria of patients with chronic AF compared with individuals in sinus rhythm. To dissect the molecular mechanism linking RyR2 oxidation to AF we used two murine models harboring RyR2 mutations that cause intracellular Ca2+ leak. Mice with intracellular Ca2+ leak exhibited increased atrial RyR2 oxidation, mitochondrial dysfunction, reactive oxygen species (ROS) production and AF susceptibility. Both genetic inhibition of mitochondrial ROS production and pharmacological treatment of RyR2 leakage prevented AF. Collectively, our results indicate that alterations of RyR2 and mitochondrial ROS generation form a vicious cycle in the development of AF. Targeting this previously unrecognized mechanism could be useful in developing effective interventions to prevent and treat AF.

In a previous report we demonstrated that RyR2 is oxidized in atrial myocytes from a murine model of CPVT, RyR2-R2474S +/mice, that display substantial increased AF susceptibility 17 .
In the present study, we explored the mechanistic role of atrial RyR2 oxidation in the pathophysiology of AF in two murine models of RyR2-mediated intracellular Ca 2+ leak: mice harboring an RyR2 mutation linked to human CPVT (RyR2-R2474S +/-) and mice expressing a phosphomimetic aspartic acid residue at position 2808 (RyR2-S2808D +/+ ) leading to constitutively leaky channels. Moreover, we evaluated the role of mitochondrial free radicals on RyR2 oxidation by crossing mice harboring RyR2 mutations associated with Ca 2+ leak with mice overexpressing human catalase targeted to mitochondria (mCAT mice).

Results
Atrial RyR2 Oxidation and Leak in Atrial Fibrillation. Atrial RyR2s from patients with chronic AF were oxidized, phosphorylated and depleted of calstabin 2 (a subunit of the complex that stabilizes the closed state of RyR2 during diastole 14,31 ) compared to subjects in sinus rhythm (Fig. 1a-d). These results are consistent with our previous report in mice harboring CPVT-mutated RyR2 channels, where RyR2 channels were oxidized and DTT treatment reduced SR Ca 2+ leak in atrial myocytes 17 .
To explore the role of intracellular Ca 2+ leak via RyR2 in the development of AF, we used a transgenic mouse harboring a constitutively leaky RyR2 channel (RyR2-S2808D +/+ ), which displays an age-related RyR2 oxidation in ventricular myocytes 32 . WT and RyR2-S2808D +/+ littermates were divided into three different age groups (3-, 6-and 9-month old). Similar to ventricular RyR2, atrial RyR2 from RyR2-S2808D +/+ mice exhibited age-related oxidation and depletion of calstabin 2 compared to RyR2 To determine RyR2 channel oxidation, the carbonyl groups in the protein side chains of immunoprecipitated RyR2 were derivatized to (DNP) by reaction with 2,4-dinitrophenylhydrazine. The DNP (2,4-dinitrophenylhydrazone) signal associated with RyR2 was determined by anti-DNP antibody. (b-d), Quantification of DNP signal (b), PKA hyperphosphorylation (c), and calstabin 2 bound to RyR2 (d) in human atrial samples. *p < 0.01 vs control. Error bars represent s.e.m. (e) Post-translational modifications of the RyR2 complex in atrial samples from WT and RyR2-S2808D +/+ mice. (f) and (g), Quantification of DNP signal (f) and calstabin 2 bound to RyR2 (g); atrial samples were obtained from at least 5 mice in each group. AU: arbitrary units. All data are shown as mean ± s.e.m. * and **: p < 0.05 and 0.01 vs 3-month-old group; # : p < 0.05 vs WT.
AF has also been associated with structural remodeling of the atria 4,6,7,9 . However, histological analyses of atrial tissue did not show any obvious structural abnormality in 9-month-old RyR2-S2808D +/+ mice (Supplementary Figure 6), suggesting that the altered Ca 2+ homeostasis is the main factor contributing to AF in this mouse model. Moreover, a 2-week pharmacological treatment with a stabilizer 14,18 of the closed state of RyR2 channel (S107, 40 mg/kg/d in drinking water) prevented the development of AF in 9-month-old RyR2-S2808D +/+ mice (Supplementary Figure 7).

Genetic inhibition of mitochondrial ROS production reduces AF.
To further investigate the role of mitochondrial ROS in atrial RyR2 dysfunction we crossed RyR2-S2808D +/+ mice with transgenic mice (mCAT) overexpressing the human catalase gene targeted to mitochondria to decrease mitochondrial ROS production. RyR2-S2808D +/+ /mCAT mice exhibited significantly reduced atrial mitochondrial abnormalities (Fig. 4a-c). In atrial myocytes isolated from 9-month old RyR2-S2808D +/+ /mCAT mice, both cellular and mitochondrial oxidative stress were markedly reduced compared with age-matched RyR2-S2808D +/+ littermates (Fig. 4d-e). Strikingly, blunted ROS production was associated with reduced atrial RyR2 oxidation and castabin2 dissociation resulting in decreased atrial diastolic SR Ca 2+ leak and AF susceptibility (Fig. 4f-h and Supplementary Figure 8-9). S107 improves mitochondrial function. To test the hypothesis that atrial mitochondrial dysfunction is attributable to SR Ca 2+ leak, we examined the effects of S107 treatment on atrial mitochondria of RyR2-S2808D +/+ mice. Following a 2-month treatment with S107 (40 mg/kg/d in the drinking water), both mitochondrial dysmorphology (Fig. 5a-c) and ROS production (Fig. 5d) were significantly reduced in 9-month-old RyR2-S2808D +/+ mice compared with vehicle group.

Discussion
Accumulating evidence suggests that oxidative stress plays a pivotal role in the development and perpetuation of AF [20][21][22]38,39 . Our present findings strongly support previous reports suggesting that AF is associated with myocardial oxidative stress 20,38 . (c) Morphometric analyses of atrial mitochondria show attenuated abnormalities in S107 treated group. (d) mitochondrial ROS levels in atrial myocytes from 9-month-old RyR2-S2808D +/+ mice following a 2-month treatment with S107 or vehicle. n > 100 cells from each group, the mitoSOX fluorescence are normalized to fold of S107 group. All data are shown as mean ± s.e.m. * and **: p < 0.05 and 0.01 vs vehicle.
Scientific RepoRts | 5:11427 | DOi: 10.1038/srep11427 We and others have proposed that atrial diastolic SR Ca 2+ leak is an essential contributing factor in the pathogenesis of AF [17][18][19] . Diastolic SR Ca 2+ leak due to RyR2 dysfunction may be linked to multiple factors including phosphorylation, oxidation or pathological mutations of RyR2, all of which can result in channel dysfunction 14 . We have previously reported PKA hyperphosphorylation and calstabin2 dissociation from RyR2 in several models of chronic AF 15 . We have also demonstrated that chronic PKA phosphorylation of ventricular RyR2 is associated with oxidation of the channel and the combination of PKA phosphorylation and oxidation of RyR2 results in significantly more calstabin 2 dissociation from RyR2 compared to each post-translational modification alone 27 . This observation is consistent with our present results, indicating the importance of oxidation of atrial RyR2 in promoting AF. Furthermore, oxidation of atrial RyR2 has been shown to be a key contributor to diastolic SR Ca 2+ leak and AF in animal models of CPVT 17 .
It has been reported that CaMKII is also a molecular target of oxidative stress, and that oxidized CaMKII can phosphorylate RyR2 at Ser 2814 inducing intracellular Ca 2+ leak 40 . However, we found that KN-93 treatment did not reduce atrial SR Ca 2+ leak in RyR2-S2808D +/+ or RyR2-R2474S +/− atrial cardiomyocytes, suggesting that direct oxidation of atrial RyR2, but not phosphorylation by oxidized CaMKII, is the main factor inducing atrial SR Ca 2+ leak and AF.
The importance of PKA phosphorylation in cardiac disease has been challenged by Valdivia's group, who concluded that phosphorylation of Ser 2808 plays no role in β -adrenergic cardiac response. The fact that our findings have been confirmed by multiple other groups is addressed in a recent review 32 .
Multiple sources of ROS, including mitochondria, NADPH oxidases and NOS uncoupling, contribute to AF 5,8,41 . Intriguingly, recent reports indicate that atrial sources of ROS vary with the duration and the substrate of AF 42 , and mitochondria have been proposed as the major ROS source for long-term AF and age-related functional decline 25 . This view is consistent with our results showing that mitochondrial ROS promote age-related AF.
In the present study we establish for the first time that atrial RyR2 is a specific molecular target of oxidative stress that is fundamental in the development of AF. We demonstrate the functional importance of RyR2 oxidation in AF pathophysiology, showing that mitochondrial-derived ROS oxidize RyR2 in atrial myocytes leading to increased intracellular Ca 2+ leak. Importantly, reducing mitochondrial ROS production attenuates atrial diastolic SR Ca 2+ leak and prevents AF.
Mitochondria have been reported to be abnormal in the atrial tissue of patients with AF 39,43 . In this study, mice harboring leaky RyR2 also display mitochondrial abnormalities and increased ROS production. In cardiac myocytes, mitochondria and the SR are co-localized in the 'mitochondrial microdomain 44 ' . Since mitochondrial Ca 2+ uptake via the mitochondrial Ca 2+ uniporter is dependent on SR Ca 2+ release [44][45][46] , alterations in SR Ca 2+ release can also affect mitochondrial function by regulating mitochondrial Ca 2+ uptake 44,47 . In atrial myocytes isolated from RyR2-S2808D +/+ mice, leaky RyR2 channels were associated with an age-related increase in diastolic SR Ca 2+ release without any significant change in the systolic Ca 2+ transient amplitudes. Pharmacological inhibition of RyR2 Ca 2+ leak restored atrial mitochondrial morphology and function suggesting that mitochondrial Ca 2+ overload plays a key role in AF pathophysiology.
Taken together, our data demonstrate that RyR2 oxidation resulting from intracellular oxidative stress in atrial myocytes leads to increased SR Ca 2+ leak contributing to the pathogenesis of AF. Alterations of RyR2 inducing intracellular Ca 2+ leak, including constitutive PKA phosphorylation or CPVT mutations, trigger a vicious cycle, in which SR Ca 2+ leak in atrial myocytes impairs mitochondrial function leading to an increase in ROS production, thereby promoting RyR2 oxidation and further Ca 2+ leak. Pharmacological targeting of leaky RyR2 channels or genetically inhibiting mitochondrial ROS production prevents AF providing mechanistic insights that could lead to new therapeutic targets for AF.

Methods
Human studies. All human analyses were performed in accordance with protocols approved by the Institutional Review Board of the New York Presbyterian Hospital and by the Ethics Committee of Columbia University. Written informed consent was obtained from all participants. RA appendage tissue was obtained at the time of cardiac surgery from patients with chronic AF (> 6 months; n = 10), and patients in sinus rhythm (n = 10).
Animal studies. All animal experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
Generation of the mCAT mouse has been described previously 48 . RyR2-R2474S +/and RyR2-S2808D +/+ mice were generated as described 17,45 . All mice were backcrossed into the C57BL/6 background for > 10 generations. All in vivo and in vitro experiments were conducted by operators who were blinded to the genotypes of the mice. S107 treatment. For in vitro experiments using isolated atrial myocytes, 10 μ M S107 was added to the extracellular solution for 2 hours. For in vivo experiments, S107 was diluted in drinking water at 0.25 mg/ml (40 mg/kg/d). No differences were detected in water consumption between vehicle and S107-treated groups.
Intra-esophageal burst pacing in mouse. Intra-esophageal pacing was performed by placing in the esophagus, close to the left atrium, a 1.1-Fr octapolar catheter (EPR-800, Millar Instruments, Houston, Texas) connected to an external stimulator (STG-3008, MultiChannel Systems, Reutlingen, Germany). A computerized data acquisition system (EMKA Technologies, Falls Church, VA) was used to record a 3-lead surface ECG, and up to 4 intra-esophageal bipolar electrocardiograms. Inducibility of atrial arrhythmias was tested by applying a series of 2-second bursts. The first 2-second burst had a cycle length (CL) of 40 ms; then the CL was progressively decreased by 2 ms in each successive burst until reaching 10 ms. AF was defined as a period of rapid irregular atrial rhythm lasting at least 1 sec.

Isolation of adult murine atrial myocytes.
Adult murine atrial myocytes were isolated as follows. The heart was rapidly isolated, cannulated and perfused with AfCS perfusion buffer, comprised of (in mM): NaCl 113, KCl 4.7, KH 2 PO 4 0.6, Na 2 HPO 4 0.6, MgSO 4 1.2, NaHCO 3 12, KHCO 3 49 . The excitation for Fluo-4 was 488 nm, while emission was collected at 505-530 nm. For Ca 2+ sparks recording, cells were scanned at 400 Hz for 20 s following 1 minute of pacing at 3 Hz. Ca 2+ sparks detection and analysis was performed as previously described 49 of Ca 2+ transient amplitudes and SR Ca 2+ contents, cells were exposed to 10 mM caffeine immediately following termination of pacing at 1 Hz for 1 minute. Sampling started 10 s before caffeine treatment.

Measurements of intracellular oxidative stress.
For the evaluation of intracellular oxidative stress, cells were pre-incubated with the chloromethyl derivative CM-H 2 DCFDA (10 μ M, ThermoFisher Scientific) for 30 minutes and washed. Fluorescence intensity and images were obtained using a confocal microscope (Zeiss 5 Live, 40 x oil immersion lens). Excitation was at 488 nm, and emission was collected at 505-530 nm. Since CM-H 2 DCFDA is light sensitive and oxidized progressively, we used the same scanning parameters for all experiments. For each dish, images were rapidly acquired for ~10 randomly selected cells. More than 100 cells per group were examined for intracellular fluorescent intensities.
Mitochondrial ROS detection. For mitochondrial ROS detection, cells were incubated for 20 min with 5 μ M MitoSOX Red (Invitrogen/Molecular probes) and washed. Fluorescence intensity and images were obtained using confocal microscopy (Zeiss 5 Live, 40x oil immersion lens). MitoSOX Red was excited at 488 nm and emission was collected at 540-625 nm. The scanning parameters were unchanged for all the scans. For each group, fluorescence intensities of > 100 cells randomly selected from several different dishes were examined.
Transmission Electron Microscopy. Atria were fixed in 2.5% glutaraldehyde in 0.1 M Sørensen's buffer and post-fixed in 1% OsO 4 . Following dehydration, samples were embedded in Lx-112 (Ladd Research Industries, Williston, VT). After cutting (ultramicrotome MT-7000), the 60 nm sections were stained with uranyl-acetate and lead-citrate and visualized (JEM-1200 EXII, JEOL, Tokyo, Japan), as previously described 45,49 . For each animal at least twenty randomly selected sections were used for the analysis of mitochondrial morphology. Statistical analysis. All results are presented as mean ± s.e.m. Statistical analyses were performed using the unpaired Student's t test (for 2 groups) and one-way ANOVA with Bonferroni post hoc correction (for groups of 3 or more) unless otherwise indicated. P < 0.05 was considered to be statistically significant.